Image-enabled cell sorting permits prospective isolation of primitive hematopoietic stem cell subsets with distinct...

Area of Science:

• Hematology
• Cell Biology
• Biotechnology

Background:

• Fluorescence activated cell sorting (FACS) is crucial for identifying and isolating hematopoietic stem cells (HSCs).
• Limitations exist in isolating HSCs based solely on marker intensity levels.

Purpose of the Study:

• To explore image-enabled cell sorting (ICS) for isolating functionally heterogeneous HSCs.
• To identify intracellular markers and parameters for prospective HSC isolation using ICS.

Main Methods:

• Utilized ICS on primary HSCs to analyze intracellular marker expression patterns.
• Integrated index sort data with single-cell divisional kinetics.
• Employed mitochondrial dyes (MitoTracker) to assess intensity and diffusivity.

Main Results:

• Identified MitoTracker dye intensity and diffusivity as key parameters for isolating distinct HSC subpopulations.
• Demonstrated enrichment of long-term multilineage HSCs in low diffusivity, low intensity MitoTracker populations.
• Discovered a novel HSC subpopulation (Mito±) isolatable only by ICS, exhibiting characteristics of low intensity populations.

Conclusions:

• ICS is a powerful tool for prospectively enriching specific cell populations within the hematopoietic system.
• ICS enables isolation of functionally heterogeneous HSCs based on intracellular marker distribution.
• This technology is broadly applicable for applications requiring high purity and precise cellular characterization.